During development of the mammalian nervous system, differentiating neurons from the central and peripheral nervous systems send out axons that must grow and make contact with specific target cells In some cases, neurons stay confined entirely within the central nervous system. In other cases, however, growing axons must cover enormous distances, extending from the CNS into the periphery of the body. In mammals, this stage of neurogenesis is completed during the embryonic phase of life and, it is believed, neuronal cells do not multiply once they have fully differentiated.
Dendritic growth occurs in two phases: initial extension followed by elongation and ramification. Purves et a/, Nature 336:123-128 (1988). Some molecules, including neurotransmitters and hormones, have been shown to regulate expansion of an existing dendritic arbor. Much less is known, however, about the factors that influence early events, and cause a neuron to initially form dendrites. In certain classes of neurons, initial dendritic sprouting occurs as part of an intrinsic developmental program which is relatively independent of trophic interactions. Dotti et a., J. Neurosci. 8:1454-1468 (1988). In other classes of neurons, however, the regulation of the initial stages of dendritic growth appears to be quite different. For example, rat sympathetic neurons fail to form dendrites and extend only axons when they are cultured in the absence of non-neuronal cells. In contrast, co-culture with Schwann cells or astrocytes causes these neurons to form dendritic processes and to eventually generate a dendritic arbor which is comparable in size to that observed in situ. Tropea et al., Glia 1:380-392 (1988). Thus, it would appear that specific trophic interactions are required to allow sympathetic neurons to form dendrites.
The foregoing observations have been taken to support a theory that the in situ environment specifies formation of a dendritic arbor. The environment in the vicinity of neural cells or developing neural processes is thought to include electromagnetic, electrochemical and/or biochemical fields or gradients which positively and negatively influence the extent and specificity of dendritic outgrowth as well as the formation of synapses between dendrites and nerve cell bodies and axons. This theory, however, suffers from a paucity of identified mediators which have the capacity to cause neurons to sprout dendrites.
A host of neuropathies, some of which affect only a subpopulation or a system of neurons in the peripheral or central nervous systems have been identified. The neuropathies, which may affect the neurons themselves or the associated glial cells, may result from cellular metabolic dysfunction, infection, exposure to toxic agents, autoimmunity dysfunction, malnutrition or ischemia. In some cases the cellular dysfunction is thought to induce cell death directly by apoptosis. Oppenheim, Ann Rev. Neurosci. 14:37-43 (1991). In other cases, the neuropathy may induce tissue necrosis by stimulating the body's immune system, resulting in a local inflammatory response and cell analysis at the initial site of neural injury.
The ability of neurons within the peripheral nervous system to regenerate a damaged neural pathway is limited. Specifically, new axons and dendrites extend randomly, and are often misdirected, making contact with inappropriate targets that can cause abnormal function. In addition, where severed nerve processes result in a gap of longer than a few millimeters, e.g., greater than 10 millimeters (mm), appropriate nerve regeneration does not occur, either because the processes fail to grow the necessary distance, or because of misdirected axonal growth. Efforts to repair peripheral nerve damage by surgical means has met with mixed results, particularly where damage extends over a significant distance. In some cases, the suturing steps used to obtain proper alignment of severed nerve ends stimulates the formulation of scar tissue which is thought to inhibit axon regeneration. Even where scar tissue formation has been reduced, as with the use of nerve guidance channels or other tubular prostheses, successful regeneration generally is still limited to nerve damage of less than 10 millimeters in distance.
It is now well established that various trophic factors play a critical role in regulating the survival and differentiation of developing neurons. Snider et al., Ann. Neurol. 26:489-506 (1989). Most of the characterized actions of nerve trophic actors relate to developmental events and suggest that the temporal and local regulation of expression of these proteins plays a role during maturation of the nervous system. Nerve trophic factors are also important in the function of the adult nervous system for the maintenance of structural integrity and regulation of plasticity. Such processes are altered in neurodegenerative diseases and neurodegenerative events following acute injury to the nervous system. This has prompted speculation that nerve trophic factors are involved in the structural alterations which occur in response to injury and disease.
Several well-characterized trophic factors have been shown to enhance the survival and differentiation of dopaminergic neurons in tissue culture and/or following transplantation to the anterior chamber of the eye. These trophic factors include fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-α (TGF-α), and glial cell-derived neurotrophic factor (GDNF), as well as several Nerve Growth Factor (NGF) related neurotrophins.
Nerve trophic factors are found among several protein superfamilies of polypeptide growth factors based on their amino acid sequence homology and/or their three-dimensional structure. MacDonald et al, Cell 73:421-424 (1993). One family of neurotrophic factors is the neurotrophin family. This family currently consists of Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4/5 (NT-4/5), and Neurotrophin-6 (NT-6). These neurotrophic factors affect specific neuronal populations in the central nervous system. The loss of such specific neurotrophic factors may be responsible for age-related declines in cell survival and/or function. While the cellular source remains unclear, there is evidence to suggest that neurons and glial cells are both capable of secreting neurotrophic factors.
The osteogenic protein/bone morphogenetic protein (OP/BMP) proteins form a family, or subfamily, within the larger TGF-β superfamily of proteins. That is, these proteins form a distinct subgroup, referred to herein as the “OP/BMP family of morphogens” or “OP/BMP morphogens,” within the loose evolutionary grouping of sequence-related proteins known as the TGF-β superfamily. Members of this protein family comprise secreted polypeptides that share common structural features, and that are similarly processed from a pro-protein to yield a carboxy-terminal mature protein. OP/BMP morphogens have been identified in developing and adult rat brain and spinal cord tissue, as determined both by northern blot hybridization of morphogen-specific probes to mRNA extracts from developing and adult nerve tissue and by immunolocalization studies. For example, northern blot analysis of developing rat tissue has identified significant OP-1mRNA transcript expression in the CNS. The mRNA of another OP/BMP family member. GDF-1, appears to be expressed primarily in developing and adult nerve tissue, specifically in the brain, including the cerebellum and brain stem, spinal cord and peripheral nerves. BMP4 (also referred to as BMP2B) and Vgr-1 transcripts also have been reported to be expressed in nerve tissue.
The morphogen OP-1 was found to be localized predominantly to the extracellular matrix of the grey matter (neuronal cell bodies), distinctly present in all areas except the cell bodies themselves. In white matter, formed mainly of myelinated nerve fibers, staining appears to be confined to astrocytes (glial cells). A similar staining pattern also was seen in newborn rat (10 day old) brain sections.
In addition, OP-1 has been specifically localized in the substantia nigra, which is composed primarily of striatal basal ganglia, a system of accessory motor neurons whose function is associated with the cerebral cortex and corticospinal system. Dysfunctions in this subpopulation or system of neurons are associated with a number of neuropathies, including Huntington's chorea and Parkinson's disease.